Cluster-Assembled Zirconia Substrates Accelerate the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Due to their high mechanical strength and good biocompatibility, nanostructured zirconia surfaces (ns-ZrOx) are widely used for bio-applications. Through supersonic cluster beam deposition, we produced ZrOx films with controllable roughness at the nanoscale, mimicking the morphological and topographical properties of the extracellular matrix. We show that a 20 nm ns-ZrOx surface accelerates the osteogenic differentiation of human bone marrow-derived MSCs (bMSCs) by increasing the deposition of calcium in the extracellular matrix and upregulating some osteogenic differentiation markers. bMSCs seeded on 20 nm ns-ZrOx show randomly oriented actin fibers, changes in nuclear morphology, and a reduction in mitochondrial transmembrane potential when compared to the cells cultured on flat zirconia (flat-ZrO2) substrates and glass coverslips used as controls. Additionally, an increase in ROS, known to promote osteogenesis, was detected after 24 h of culture on 20 nm ns-ZrOx. All the modifications induced by the ns-ZrOx surface are rescued after the first hours of culture. We propose that ns-ZrOx-induced cytoskeletal remodeling transmits signals generated by the extracellular environment to the nucleus, with the consequent modulation of the expression of genes controlling cell fate.

A Novel Fluidic Platform for Semi-Automated Cell Culture into Multiwell-like Bioreactors.

In this work, we developed and characterized a novel fluidic platform that enables long-term in vitro cell culture in a semi-automated fashion. The system is constituted by a control unit provided with a piezoelectric pump, miniaturized valves, and a microfluidic network for management and fine control of reagents' flow, connected to a disposable polymeric culture unit resembling the traditional multiwell-like design. As a proof of principle, Human Umbilical Vein Endothelial Cells (HUVEC) and Human Mesenchymal Stem Cells (hMSC) were seeded and cultured into the cell culture unit. The proliferation rate of HUVEC and the osteogenic differentiation of hMSC were assessed and compared to standard culture in Petri dishes. The results obtained demonstrated that our approach is suitable to perform semi-automated cell culture protocols, minimizing the contribution of human operators and allowing the standardization and reproducibility of the procedures. We believe that the proposed system constitutes a promising solution for the realization of user-friendly automated control systems that will favor the standardization of cell culture processes for cell factories, drug testing, and biomedical research.

HeLLePhant: A phantom mimicking non-small cell lung cancer for texture analysis in CT images.

PURPOSE: Phantoms mimicking human tissue heterogeneity and intensity are required to establish radiomic features robustness in Computed Tomography (CT) images. We developed inserts with two different techniques for the radiomic study of Non-Small Cell Lung Cancer (NSCLC) lesions. METHODS: We developed two insert prototypes: two 3D-printed made of glycol-modified polyethylene terephthalate (PET-G), and nine with sodium polyacrylate plus iodinated contrast medium. The inserts were put in a handcraft phantom (HeLLePhant). We also analysed four materials of a commercial homogeneous phantom (Catphan® 424) and collected 29 NSCLC patients for comparison. All the CT acquisitions were performed with the same clinical protocol and scanner at 120kVp. The HeLLePhant phantom was scanned ten times in fixed condition at 120kVp and 100kVp for repeatability investigation. We extracted 153 radiomic features using Pyradiomics. To compare the features between phantoms and patients, we computed how many phantom features fell in the range between 10th and 90th percentile of the corresponding patient values. We deemed repeatable the features with a coefficient of variation (CV) less than or equal to 0.10. RESULTS: The best similarity with the patients was obtained with the polyacrylate inserts (55.6-90.2%), the worst with Catphan (15.7-19.0%). For the PET-G inserts 35.3% and 36.6% of the features match the patient range. We found high repeatability for all the inserts of the HeLLePhant phantom (74.3-100% at 120kVp, 75.7-97.9% at 100kVp), and observed a texture dependency in repeatability. CONCLUSIONS: Our study shows a promising way to construct heterogeneous inserts mimicking a target tissue for radiomic studies.

Monolithic Three-Dimensional Functionally Graded Hydrogels for Bioinspired Soft Robots Fabrication.

Bioinspired soft robotics aims at reproducing the complex hierarchy and architecture of biological tissues within artificial systems to achieve the typical motility and adaptability of living organisms. The development of suitable fabrication approaches to produce monolithic bodies provided with embedded variable morphological and mechanical properties, typically encountered in nature, is still a technological challenge. Here we report on a novel manufacturing approach to produce three-dimensional functionally graded hydrogels (3D-FGHs) provided with a controlled porosity gradient conferring them variable stiffness. 3D-FGHs are fabricated by means of a custom-designed liquid foam templating (LFT) technique, which relies on the inclusion of air bubbles generated by a blowing agent into the monomer-based template solution during ultraviolet-induced photopolymerization. The 3D-FGHs' apparent Young's modulus ranges from 0.37 MPa (bulky hydrogel region) to 0.09 MPa (highest porosity region). A fish-shaped soft swimmer is fabricated to demonstrate the feasibility of the LFT technique to produce bioinspired robots. Mobility tests show a significant improvement in terms of swimming speed when the robot is provided with a graded body. The proposed manufacturing approach constitutes an enabling solution for the development of macroscopic functionally graded hydrogel-based structures usable in biomimetic underwater soft robotics applications.

Effects of the 3D Geometry Reconstruction on the Estimation of 3D Porous Scaffold Permeability.

3D scaffolds for tissue engineering typically need to adopt a dynamic culture to foster cell distribution and survival throughout the scaffold. It is, therefore, crucial to know fluids' behavior inside the scaffold architecture, especially for complex porous ones. Here we report a comparison between simulated and measured permeability of a porous 3D scaffold, focusing on different modeling parameters. The scaffold features were extracted by microcomputed tomography (µCT) and representative volume elements were used for the computational fluid-dynamic analyses. The objective was to investigate the sensitivity of the model to the degree of detail of the µCT image and the elements of the mesh. These findings highlight the pros and cons of the modeling strategy adopted and the importance of such parameters in analyzing fluid behavior in 3D scaffolds.

Hybrid Additive Fabrication of a Transparent Liver with Biosimilar Haptic Response for Preoperative Planning.

Due to the complexity of liver surgery, the interest in 3D printing is constantly increasing among hepatobiliary surgeons. The aim of this study was to produce a patient-specific transparent life-sized liver model with tissue-like haptic properties by combining additive manufacturing and 3D moulding. A multistep pipeline was adopted to obtain accurate 3D printable models. Semiautomatic segmentation and registration of routine medical imaging using 3D Slicer software allowed to obtain digital objects representing the structures of interest (liver parenchyma, vasculo-biliary branching, and intrahepatic lesion). The virtual models were used as the source data for a hybrid fabrication process based on additive manufacturing using soft resins and casting of tissue-mimicking silicone-based blend into 3D moulds. The model of the haptic liver reproduced with high fidelity the vasculo-biliary branching and the relationship with the intrahepatic lesion embedded into the transparent parenchyma. It offered high-quality haptic perception and a remarkable degree of surgical and anatomical information. Our 3D transparent model with haptic properties can help surgeons understand the spatial changes of intrahepatic structures during surgical manoeuvres, optimising preoperative surgical planning.

Direct 3D Printing of Clear Orthodontic Aligners: Current State and Future Possibilities.

The recent introduction of three-dimensional (3D) printing is revolutionizing dentistry and is even being applied to orthodontic treatment of malocclusion. Clear, personalized, removable aligners are a suitable alternative to conventional orthodontic appliances, offering a more comfortable and efficient solution for patients. Including improved oral hygiene and aesthetics during treatment. Contemporarily, clear aligners are produced by a thermoforming process using various types of thermoplastic materials. The thermoforming procedure alters the properties of the material, and the intraoral environment further modifies the properties of a clear aligner, affecting overall performance of the material. Direct 3D printing offers the creation of highly precise clear aligners with soft edges, digitally designed and identically reproduced for an entire set of treatment aligners; offering a better fit, higher efficacy, and reproducibility. Despite the known benefits of 3D printing and the popularity of its dental applications, very limited technical and clinical data are available in the literature about directly printed clear aligners. The present article discusses the advantages of 3D printed aligners in comparison to thermoformed ones, describes the current state of the art, including a discussion of the possible road blocks that exist such as a current lack of approved and marketed materials and limited existence of aligner specific software. The present review suggests the suitability of 3D direct printed aligners is superior to that of thermoformed manufactured aligners because of the prior's increased accuracy, load resistance, and lower deformation. It is an overall more stable way to produce an aligner where submillimeter movements can make a difference in treatment outcome. Direct 3D printing represents a complex method to control the thickness of the aligner and therefore has a better ability to control the force vectors that are used to produce tooth movement. There is currently no other approved material on the market that can do this. The conclusion of this article is that we encourage further in vitro and in vivo studies to test these new technologies and materials.

Additive Fabrication of a Vascular 3D Phantom for Stereotactic Radiosurgery of Arteriovenous Malformations.

In this study, an efficient methodology for manufacturing a realistic three-dimensional (3D) cerebrovascular phantom resembling a brain arteriovenous malformation (AVM) for applications in stereotactic radiosurgery is presented. The AVM vascular structure was 3D reconstructed from brain computed tomography (CT) data acquired from a patient. For the phantom fabrication, stereolithography was used to produce the AVM model and combined with silicone casting to mimic the brain parenchyma surrounding the vascular structure. This model was made with tissues-equivalent materials for radiology. The hollow vascular system of the phantom was filled with a contrast agent usually employed on patients for CT scans. The radiological response of the phantom was tested and compared with the one of the clinical case. The constructed model demonstrated to be a very accurate physical representation of the AVM and its vasculature and good morphological consistency was observed between the model and the patient-specific source anatomy. These results suggest that the proposed method has potential to be used to fabricate patient-specific phantoms for neurovascular radiosurgery applications and medical research.

Eco-Friendly Supercapacitors Based on Biodegradable Poly(3-Hydroxy-Butyrate) and Ionic Liquids.

The interest for biodegradable electronic devices is rapidly increasing for application in the field of wearable electronics, precision agriculture, biomedicine, and environmental monitoring. Energy storage devices integrated on polymeric substrates are of particular interest to enable the large-scale on field use of complex devices. This work presents a novel class of eco-friendly supercapacitors based on biodegradable poly(3-hydroxybutyrrate) PHB, ionic liquids, and cluster-assembled gold electrodes. By electrochemical characterization, we demonstrate the possibility of tuning the supercapacitor energetic performance according to the type and amount of the ionic liquid employed. Our devices based on hydrophobic plastic materials are stable under cyclic operation and resistant to moisture exposure.

Combined Effects of Electrical Stimulation and Protein Coatings on Myotube Formation in a Soft Porous Scaffold.

Compared to two-dimensional cell cultures, three-dimensional ones potentially allow recreating natural tissue environments with higher accuracy. The three-dimensional approach is being investigated in the field of tissue engineering targeting the reconstruction of various tissues, among which skeletal muscle. Skeletal muscle is an electroactive tissue which strongly relies upon interactions with the extracellular matrix for internal organization and mechanical function. Studying the optimization of myogenesis in vitro implies focusing on appropriate biomimetic stimuli, as biochemical and electrical ones. Here we present a three-dimensional polyurethane-based soft porous scaffold (porosity ~ 86%) with a Young's modulus in wet conditions close to the one of natural skeletal muscle tissue (~ 9 kPa). To study the effect of external stimuli on muscle cells, we functionalized the scaffold with extracellular matrix components (laminin and fibronectin) and observed an increase in myoblast proliferation over three days. Furthermore, the combination between laminin coating and electrical stimulation resulted in more spread and thicker myotubes compared to non-stimulated samples and samples receiving the single (non-combined) inputs. These results pave the way to the development of mature muscle tissue within three-dimensional soft scaffolds, through the combination of biochemical and electrical stimuli.

Soft Piezoionic/Piezoelectric Nanocomposites Based on Ionogel/BaTiO3 Nanoparticles for Low Frequency and Directional Discriminative Pressure Sensing.

We report on the fabrication and electro-mechanical characterization of a nanocomposite system exhibiting anisotropic electrical response under the application of tactile compressive stresses (5 kPa) at low frequencies (0.1-1 Hz). The nanocomposite is based on a chemically cross-linked gel incorporating a highly conductive ionic liquid and surface functionalized barium titanate (BaTiO3) ferroelectric nanoparticles. The system was engineered to respond to mechanical stimulations by combining piezoionic and piezoelectric activity, generating electric charge due to a redistribution of the mobile ions across the polymer matrix and to the presence of the electrically polarized ceramic nanoparticles, respectively. The nanocomposite response was characterized in a quasi-static regime using a custom-designed apparatus. The results obtained showed that the combination of both piezo-effects led to output voltages up to 8 mV and anisotropy in the response. This allows to discriminate the sample orientation with respect to the load direction by monitoring the phase and amplitude modulation of the output signal. The integration of cluster-assembled gold electrodes produced by Supersonic Cluster Beam Deposition (SCBD) was also performed, enabling to enhance the charge transduction efficiency by a factor of 10, compared to the bare nanocomposite. This smart piezoionic/piezoelectric nanocomposite represents an interesting solution for the development of soft devices for discriminative touch sensing and objects localization in physically unstructured environments.

An Amperometric Sensor for Thiocholine Based on Cluster-Assembled Zirconia Modified Electrodes.

Here we report on the fabrication and characterization of cluster-assembled nanostructured zirconia electrodes for the electrochemical detection of enzymatically produced thiocholine. Zirconia nanostructures are produced by Supersonic Cluster Beam Deposition on thin gold films. This technique enables nanoscale control of the deposited film surface morphology, providing high active surface area for electrochemical detection of the analyte, along with high double-layer capacitance and suitable charge transfer resistance of the system. The electrochemical behavior of the electrodes has been characterized in the presence of the Potassium ferricyanide/Potassium ferrocyanide redox couple and the system performance showed to be enhanced starting from a thickness of the deposited layer of 60 nm. The electrochemical response for the oxidation of an enzymatic product was assessed by means of cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry. The nanostructured zirconia film yields a good electrochemical detection of thiocholine. The limit of detection for thiocholine under working potential of 0.810-0.820 V versus reference was found to be comprised between 0.25 µM and 1.3 µM. Nanostructured electrodes, combining gold and zirconia nanoparticles can be implemented as functional transducers in biosensing devices, for example based on Acetylcholinesterase for electrochemical detection of polluting agents.

3D porous polyurethanes featured by different mechanical properties: Characterization and interaction with skeletal muscle cells.

The fabrication of biomaterials for interaction with muscle cells has attracted significant interest in the last decades. However, 3D porous scaffolds featured by a relatively low stiffness (almost matching the natural muscle one) and highly stable in response to cyclic loadings are not available at present, in this context. This work describes 3D polyurethane-based porous scaffolds featured by different mechanical properties. Biomaterial stiffness was finely tuned by varying the cross-linking degree of the starting foam. Compression tests revealed, for the softest material formulation, stiffness values close to the ones possessed by natural skeletal muscles. The materials were also characterized in terms of local nanoindenting, rheometric properties and long-term stability through cyclic compressions, in a strain range reflecting the contraction extent of natural muscles. Preliminary in vitro tests revealed a preferential adhesion of C2C12 skeletal muscle cells over the softer, rougher and more porous structures. All the material formulations showed low cytotoxicity.

Electroactive Ionic Soft Actuators with Monolithically Integrated Gold Nanocomposite Electrodes.

Electroactive ionic gel/metal nanocomposites are produced by implanting supersonically accelerated neutral gold nanoparticles into a novel chemically crosslinked ion conductive soft polymer. The ionic gel consists of chemically crosslinked poly(acrylic acid) and polyacrylonitrile networks, blended with halloysite nanoclays and imidazolium-based ionic liquid. The material exhibits mechanical properties similar to that of elastomers (Young's modulus ≈ 0.35 MPa) together with high ionic conductivity. The fabrication of thin (≈100 nm thick) nanostructured compliant electrodes by means of supersonic cluster beam implantation (SCBI) does not significantly alter the mechanical properties of the soft polymer and provides controlled electrical properties and large surface area for ions storage. SCBI is cost effective and suitable for the scaleup manufacturing of electroactive soft actuators. This study reports the high-strain electromechanical actuation performance of the novel ionic gel/metal nanocomposites in a low-voltage regime (from 0.1 to 5 V), with long-term stability up to 76 000 cycles with no electrode delamination or deterioration. The observed behavior is due to both the intrinsic features of the ionic gel (elasticity and ionic transport capability) and the electrical and morphological features of the electrodes, providing low specific resistance (<100 Ω cm-2 ), high electrochemical capacitance (≈mF g-1 ), and minimal mechanical stress at the polymer/metal composite interface upon deformation.